MDM2 inhibitors, nutlin-3a and navtemadelin, retain efficacy in human and mouse cancer cells cultured in hypoxia

Activation of p53 by small molecule MDM2 inhibitors can induce cell cycle arrest or death in p53 wildtype cancer cells. However, cancer cells exposed to hypoxia can develop resistance to other small molecules, such as chemotherapies, that activate p53. Here, we evaluated whether hypoxia could render cancer cells insensitive to two MDM2 inhibitors with different potencies, nutlin-3a and navtemadlin. Inhibitor efficacy and potency were evaluated under short-term hypoxic conditions in human and mouse cancer cells expressing different p53 genotypes (wild-type, mutant, or null). Treatment of wild-type p53 cancer cells with MDM2 inhibitors reduced cell growth by > 75% in hypoxia through activation of the p53–p21 signaling pathway; no inhibitor-induced growth reduction was observed in hypoxic mutant or null p53 cells except at very high concentrations. The concentration of inhibitors needed to induce the maximal p53 response was not significantly different in hypoxia compared to normoxia. However, inhibitor efficacy varied by species and by cell line, with stronger effects at lower concentrations observed in human cell lines than in mouse cell lines grown as 2D and 3D cultures. Together, these results indicate that MDM2 inhibitors retain efficacy in hypoxia, suggesting they could be useful for targeting acutely hypoxic cancer cells.

The tumor suppressor protein p53 regulates the cellular response to different types of stress. Under normal (stress-free) conditions, p53 is expressed at a basal level, due to its ubiquitination and proteasomal degradation mediated by the E3 ligases MDM2 and MDMX. However, under stressed conditions, p53 is stabilized and functionally activated, leading to the transactivation of target genes involved in DNA repair, senescence, cell cycle, and/or apoptosis 1 . Given that this response protects cells from damage that can lead to tumor formation, it is not surprising that the TP53 gene is inactivated or mutated in roughly 50% of all cancers 2 . In the remaining cancers with wildtype p53, protein expression can be silenced through upstream alterations. Restoration of wildtype p53 activity in tumors is therefore an important goal for improved cancer treatment 3 .
Several small molecules have been developed to restore p53 activity in cancer cells expressing wildtype p53. One class of these molecules, the MDM2 inhibitors, prevent the interaction between p53 and its negative regulator MDM2. As a result, p53 accumulates in the cell and activates its downstream target genes 4 . While treatment with inhibitors alone tends to induce cell cycle arrest, treatment with inhibitors combined with chemotherapy or radiation tends to enhance apoptosis in vitro and in preclinical tumor models [5][6][7][8][9][10] , although with varying potencies. For example, one of the most studied MDM2 inhibitors, nutlin-3a, requires two to threefold higher concentrations to induce p53 than other inhibitors (e.g., navtemadlin) require 6,11,12 , and may have off-target effects [12][13][14] . Although many of these inhibitors show great promise in preclinical and clinical trials, the development of resistance is a concern 15 . Hypoxia, which commonly occurs in solid tumors 16 , can render cancer cells resistant to chemotherapies that induce their apoptotic activity through p53-dependent mechanisms 17,18 . This resistance is thought to arise partly from transcriptional and/or translational changes that cells undergo to survive hypoxia 19 . For example, upregulation of hypoxia responsive miRNAs has been shown to render colorectal cancer cells resistant to treatment with 5-fluorouracil 20 . Even 4 h exposure to hypoxia has been shown to reduce ribosome translation in cancer cells 21 , which could in turn affect p53 transcriptional activity 22 . In addition, hypoxia may select for cells that have reduced potential for p53-mediated apoptosis 23 , potentially affecting the ability of small molecules to activate a p53-mediated response. Moreover, hypoxia has been reported to reduce cell proliferation 24 , which could spare hypoxic cells from the cytotoxic effects of chemotherapies targeting proliferating cells. Since hypoxia-mediated resistance is associated with poorer clinical prognosis in several solid tumors 25 , evaluating the ability of MDM2 inhibitors to induce a p53 response in hypoxia is important for their successful clinical use.
Although many such inhibitors have been developed, few have been evaluated for their efficacy in hypoxia. One study found that the small molecule RITA, which binds directly to the N-terminus of p53, induced p53-dependent apoptosis in human colorectal cancer cell lines cultured in hypoxia 26 . However, RITA has also been reported to exert apoptosis in a non-p53 dependent manner 27 . Another study reported that the MDM2 inhibitor nutlin-3 activated p53 and inhibited growth in mouse melanoma cells cultured in hypoxia 28 . However, nutlin-3 and its more active enantiomer nutlin-3a have poor bioavailability for clinical use. To our knowledge, no studies have evaluated the efficacy of more potent and bioavailable small molecule MDM2 inhibitors currently in clinical trials, such as navtemadlin (previously known as AMG 232), in hypoxic cancer cells.
Here, we evaluated the ability of two MDM2 inhibitors with different potencies, nutlin-3a and navtemadlin, to induce p53 levels in human and mouse tumor cells cultured in hypoxia. Changes in cell proliferation were quantified using growth assays and flow cytometry, while molecular changes in the p53-p21 axis were detected using transcriptional and translational assays. Our results show that both small molecule MDM2 inhibitors, nutlin-3a and navtemadlin, induce a p53 response in hypoxic cancer cells with wildtype p53, but with varying efficacies depending on species and cell line.
Cell cycle analysis. The effect of MDM2 inhibitors on cell cycle distribution was assessed using flow cytometry. Cells (1 × 10 5 ) were seeded in T-25 culture flasks and incubated for 72 h prior to treatment. After the medium in each flask was replaced with fresh medium (5 mL/flask) containing DMSO (< 0.1%), nutlin-3a (2 µM for human cells; 10 µM for mouse cells), or navtemadlin (0.5 µM for human cells; 2 µM for mouse cells), cells were incubated either in normoxia or in hypoxia for a total of 24 h to measure molecular changes that precede changes in growth 6,7,31 . Given differences in potencies across inhibitors and across species, we used different concentrations of each inhibitor (close to the IC 50 ) in human vs mouse cell lines to achieve similar efficacies. One hour prior to trypsin-mediated harvest, cells were treated with 10 µM of EdU (5-ethynyl-2´-deoxyuridine) to mark cells in S phase. Cells were then fixed by adding 100 µL of Click-iT® fixative (Click-iT™ Plus EdU Alexa qPCR. Gene expression induced by MDM2 inhibitors in normoxia and hypoxia was measured using qPCR. Cells (1 × 10 5 cells in 2 mL/well) were seeded in a 6-well tissue culture dish (TPP) and allowed to attach for 36 h. Drug treatment was carried out by replacing the medium with fresh medium containing DMSO (< 0.1%), nutlin-3a (2 µM for human cells; 10 µM for mouse cells), or navtemadlin (0.5 µM for human cells; 2 µM for mouse cells), and incubating cells in normoxia or hypoxia for 24 h. Cells were lysed using RNA binding lysis buffer (500 µL/well; mirVana™ miRNA Isolation Kit, ThermoFisher), and lysates were stored at − 80 °C until extraction. Total RNA was extracted using the manufacturer's protocol (mirVana™ miRNA Isolation Kit, ThermoFisher Scientific or RNAeasy, Qiagen) and stored at − 20 °C. cDNA was obtained by reverse transcription (iScript cDNA Synthesis Kit, BIO RAD). qPCR was performed in duplicate in a 96-well plate according to manufacturer's instructions (iTaq Universal SYBR Green Supermix, BioRad) using pre-designed, PrimeTime qPCR primers containing SYBR Green (Integrated DNA Technologies; Table 1). Primers were confirmed to have 90-110% efficiency using the standard curve method. Gene expression was measured for two target genes (CDKN1A and MDM2 for human; Cdkn1a and Mdm2 for mouse) and reference genes (B2M for human and Rplp0 for mouse).
StepOnePlus™ Software v2.3 was used to analyze the data and fold change was calculated using the 2-Ct method, with values for target genes normalized to the reference genes.

Western blotting.
To determine whether MDM2 inhibitors induce known changes in the p53-p21 axis at the protein level in hypoxia, we examined p53, p21, MDM2 and HIF-1α levels in normoxia and hypoxia using immunoblotting. Cells (4 × 10 5 cells in 3 mL/dish) were seeded in a 60 mm tissue culture dish (TPP) and allowed to attach for 36 h. Drug treatment was carried out by replacing the medium with fresh medium containing DMSO (< 0.1%), nutlin-3a (2 µM for human cells; 10 µM for mouse cells), or navtemadlin (0.5 µM for human cells; 2 µM for mouse cells), and incubating cells in normoxia or hypoxia for 24 h. Adherent cells were washed with PBS, lysed with SDS lysis buffer (1X SDS lysis buffer, BioRad), heated at 95 °C for 5 min, sonicated (Qsonica Sonicators) for 30 s at 20% amplitude, and stored at − 20 °C until use.
Prior to electrophoresis, protein concentrations were determined using the DC Protein Assay (BioRad) according to manufacturer's instructions. Equal amounts of protein (15 µg) were loaded on 4-15% 1D polyacrylamide Mini-PROTEAN TGX Stain-Free gels (BioRad) and run in 1X Tris Glycine SDS buffer (BioRad) at 50 V for 5 min and 150 V for 45 min. Gels were activated by a 5 min exposure using an Imaging System (ChemiDoc™ Touch, BioRad). Proteins were then transferred to a Minisize PVDF membrane (BioRad) using a semi-dry transfer (TurboBlot, BioRad) for 30 min using the standard built-in protocol. Membranes were imaged using the Imaging System (ChemiDoc™ Touch, BioRad) to obtain images of total protein and subsequently equilibrated in PBS-T (0.1% Tween-20 in PBS) for 15 min. Membranes were incubated in PBS-T containing 5% milk for 1 h at room temperature to block non-specific binding. Membranes were then incubated with primary antibodies overnight at 4 °C ( Table 2). The next day, membranes were washed 4 times for 5 min in PBS-T and then incubated for 1 h room temperature with secondary antibodies ( Table 2). After membranes were washed 6 times for 5 min in PBS-T, they were incubated with ECL Clarity Substrate and Reagent (Clarity™ Western ECL Substrate, 1705060, BioRad) for 5 min and imaged for chemiluminescence signal (ChemiDoc™ Touch, BioRad).

Spheroid growth assays.
To determine whether MDM2 inhibitors could reduce growth in 3D tumors comprising innate hypoxia, we measured the growth of 3D spheroids under treatment. Single cell solutions (3 × 10 3 cells in 100 μL/well) were seeded in ultra-low attachment, round-bottom, 96-well plates to generate spheroids (Corning 7007). After the formation of spheroids (72 h), fresh medium (100 μL) containing twice the final concentration of drug was added to each well resulting in a total volume of 200 μL. Spheroids from www.nature.com/scientificreports/ human cells were treated with a final concentration of 5 μM nutlin-3a, 1 μM navtemadlin, and 2 μM staurosporine; concentrations of inhibitors were increased to limit potential drug resistance commonly observed in spheroid models [33][34][35] . Spheroids from mouse cells were treated with a final concentration of 10 μM nutlin-3a, 2 μM navtemadlin, and 5 μM staurosporine; concentrations were not increased for mouse spheroids to avoid off-target effects of nutlin-3a 13,14 and to minimize spheroid dissociation during navtemadlin treatment. Half the medium was replaced with fresh drug-containing medium every 2 days. Spheroids were imaged during treatment for up to 4-7 days (Biospa 8 or Incucyte Software). Images were analyzed using SpheroidSizer 36 .

Statistical analysis.
After checking for homogeneity of variance, data were evaluated for statistical significance using a mixed-effect, two-way ANOVA with Dunnett's multiple comparison's test (unpaired, two-tailed, α= 0.05). To avoid violating assumptions in normality, statistical significance for qPCR data was calculated on dCt values (the difference between sample and reference Ct values). For monolayer assays, data points represent the average value of technical replicates from one experiment ( ≥ 3 biological experiments). For spheroid assays, data points represent the median value ± 95% CI combined from ≥ 2 biological experiments. Randomization and blinding were not possible.
To determine whether the reduction in growth resulted from fewer proliferating cells, we then measured the uptake of 5-ethynyl-2'-deoxyuridine (a marker of DNA synthesis) in cells treated with MDM2 inhibitors for 24 h 6,31 in normoxia or hypoxia. In HCT116 p53 +/+ cells, treatment with both inhibitors in normoxia led to a > 60% decrease in S phase (nutlin-3a, P adj = 0.0016; navtemadlin, P adj < 0.0001; Fig. 2a). In hypoxia, however, only treatment with navtemadlin led to a significant decrease in S phase (nutlin-3a, P adj = 0.08; navtemadlin, P adj < 0.0001; Fig. 2a). Although hypoxia itself is reported to reduce cell proliferation and induce cell cycle arrest 24 , it alone did not affect proliferation after 24 h in HCT116 p53 +/+ cells (S phase, DMSO normoxia vs hypoxia, P adj = 0.92). No significant changes in cell proliferation were measured for HCT116 p53 −/− cell lines treated in either normoxia or hypoxia (Fig. 2b). Thus, treatment of HCT116 p53 +/+ cells with MDM2 inhibitors under hypoxia reduces cellular growth through induction of cell cycle arrest.
Nutlin-3a and navtemadlin reduce the growth of p53 WT spheroids comprising innate hypoxia. Many studies have shown that 3D cancer models mimic many features of solid tumors, including variations in oxygen levels 37,38 , gene expression 39,40 , metabolism 41 , and drug response profiles 34,39,40,42 . We therefore tested whether nutlin-3a and navtemadlin could reduce cell growth in tumor spheroids, a 3D in vitro model that naturally develops regions of hypoxia and necrosis at spheroid diameters of 400-600 μm due to an oxygen diffusion gradient 37,43,44 . To this end, we generated tumor spheroids from the p53 WT cell lines and treated them with the inhibitors when spheroids reached a diameter (> 500-550 μm). Both nutlin-3a and navtemadlin suppressed the growth of p53 WT spheroids from human cell lines (HCT116 p53 +/+ and MCF7) by > 75% when compared to DMSO. In contrast, based on volume metrics alone, nutlin-3a did not significantly reduce the growth of mouse p53 WT spheroids, while navtemadlin reduced growth by nearly 60% (Fig. 5a, b). We observed that inhibitor treatment had varying effects on spheroid intactness depending on the cell line. While nearly all HCT116 p53 +/+ spheroids shrunk from their starting size after treatment, a solid mass of ~ 450-500 μm in diameter remained (Fig. 5a). In contrast, MCF7 spheroids were primarily cell debris (Fig. 5a). B16-F10 p53 +/+ spheroids treated with nutlin-3a and navtemadlin appeared to be intact by brightfield microscopy but broke apart easily into cell debris upon pipetting. Upon close-up visualization of these B16-F10 spheroids, we noticed blebbing on the spheroid surface suggesting that cells may have been undergoing apoptosis (Fig. 5c). As expected, no www.nature.com/scientificreports/ growth suppression was observed with inhibitor treatment in p53 KO spheroids (Fig. 5d, e). Treatment with staurosporine, a non p53-specific inducer of apoptosis, prevented both p53 WT and p53 KO spheroids from growing (Fig. 5d, e). Taken together, these findings suggest that both MDM2 inhibitors reduce the growth of human and mouse p53 WT cancer spheroids comprising innate hypoxia, although their efficacy varies by cell line and species.

Discussion
Our results show that the MDM2 inhibitors, nutlin-3a and navtemadlin, induce wild-type p53 in cancer cell lines cultured in hypoxia. Contrary to what we expected, the inhibitors reduced the growth of human and mouse p53 WT cells in hypoxia through activation of the p53-p21 axis. The concentrations (IC 50 values) required to induce these effects were not significantly different between normoxic and hypoxic conditions, and were consistent with values reported in previous studies performed in normoxia 6,31 . We also found that the inhibitors reduced growth of human and mouse cancer cells grown as 3D spheroid models comprising innate hypoxia, suggesting that the inhibitors retain efficacy even when cells are hypoxic prior to treatment. Taken together, these findings indicate that the efficacy and potency of MDM2 inhibitors are not reduced in p53 WT cells cultured in hypoxia. Amongst the two inhibitors, nutlin-3a was less potent than navtemadlin at inducing a p53 response in all p53 WT cells cultured in hypoxia, consistent with results of previous studies performed in normoxia. The effect of nutlin-3a on cell growth may reflect an off-target effect and can occur independently of p53 through induction of DNA damage 13,14 . In contrast, navtemadlin has shown remarkable selectivity in human and mouse cells expressing p53 WT7,11,29,31 , almost no reported off-target effects in mouse cells 29 , and an acceptable pharmacokinetic profile in cancer patients 45 . Our current findings showing navtemadlin's efficacy in hypoxic conditions add further value to this inhibitor currently being evaluated in clinical trials.
Although the potency and efficacy of the two MDM2 inhibitors were not altered in hypoxia, their efficacy varied by species, as evidenced in three ways. First, lower concentrations of inhibitors were required to elicit a p53 response in human cells than in mouse cells used in this study. Second, treatment with the inhibitors induced transcriptional activity by more than ninefold in human cells, but by only threefold in mouse cells. Third, treatment with the inhibitors significantly reduced the volume of spheroids comprising human cells. However, they had variable effects on the volume of spheroids comprising mouse cells: nutlin-3a did not significantly reduce the volume of murine spheroids while navtemadlin did. Nevertheless, both inhibitors appeared to affect the integrity and morphology of the spheroids comprising mouse cells, suggesting that MDM2 inhibitors as monotherapy induce cell cycle arrest and moderate levels of apoptosis in mouse cells, as we have previously shown under normoxia 29 . Although we do not know the exact reason for this variation across species, we speculate that differences in expression of p14ARF (or p19ARF in mice) among the cell lines may affect the efficacy of MDM2 inhibitors. Previous studies show that p19ARF can prevent p53 degradation through inactivation of MDM2's ligase activity 46,47 . Given that the mouse B16-F10 cell line is p19ARF deficient 48 , higher concentrations of inhibitors may be required to "bind" all the free MDM2 and stabilize p53. This explanation is consistent with previous spheroids treated with DMSO, nutlin-3a, navtemadlin and staurosporine. Data points represent the median value ± 95% CI (n = 6-18 spheroids/treatment) from 2 to 3 independent experiments. Statistical significance was assessed by mixed-effects ANOVA with matching followed by post-hoc Dunnett's correction for multiple testing (unpaired, two-tailed, α = 0.05). Adjusted P values from the Dunnett's test are indicated (*P < 0.05; **P < 0.01; ***P < 0.001). www.nature.com/scientificreports/ reports showing that cell lines deficient in p19ARF are less able to elicit a p53 response 48 . Despite these species differences, our results confirm that both human and mouse cells respond to MDM2 inhibitors in hypoxia. Hypoxia alone also had varying effects by species. In the human p53 WT and p53 KO cancer cells studied here, 24 h exposure to hypoxia (1% O 2 ) itself did not measurably induce cell cycle arrest, as evidenced by the lack of p21 expression in Western blots and by the lack of differences in S phase cells in hypoxia. By contrast, the same settings of hypoxia induced cell cycle arrest in the mouse p53 WT and p53 KO cell lines, as evidenced by the drop in S phase fraction of hypoxic cells. These phenotypic differences are consistent with those reported in the literature-some cell lines arrest in hypoxia, while others do not 24,49,50 . One potential mechanistic explanation is that p14ARF/p19ARF expression may protect cells from hypoxia-induced arrest, as p14ARF has been shown to inhibit HIF-1α transcriptional activity 51 . The phenotypic differences may also arise from other variables such as the length of hypoxic exposure (ranging from 24 to 72 h), oxygen levels (ranging from 0.02 to 1.4%), and cell seeding densities that influence gene expression 24,52 .
Could MDM2 inhibitors induce p53 response in hypoxic regions of solid tumors? We found that while the inhibitors prevented the growth of 3D tumor spheroids, they did not eliminate them for some cell lines (e.g., HCT116). We hypothesize that this result could be due to cell cycle arrest or quiescence induced by hypoxia within the 3D spheroid 38,40,43 , as long periods of hypoxia have been shown to arrest cells or to induce quiescence. Solid tumors also have acute regions of hypoxia (resulting from limited blood flow) and chronic regions of hypoxia (resulting from limited oxygen perfusion) 16 . Given that p53 is transcriptionally active only in proliferating cells 53 , we expect therefore that MDM2 inhibitors would have reduced efficacy in arrested or quiescent cells. As such, we reason that the inhibitors would likely elicit a response in cancer cells experiencing acute hypoxia, but not in those experiencing chronic hypoxia. Further studies are required to dissect responses in chronic hypoxia.

Data availability
The datasets generated during the current study are included in the manuscript or available in the Zenodo repository (https:// doi. org/ 10. 5281/ zenodo. 74766 70). Any other relevant data are available upon reasonable request from the corresponding authors.